Hepatitis C virus (HCV), an example of a Flaviviridae virus, is the principal etiological agent of post-transfusion and community-acquired non-A non-B hepatitis worldwide. It is estimated that over 150 million people worldwide are infected by the virus. A high percentage of carriers become chronically infected with this pathogen and many patients progress to a state of chronic liver disease, so-called chronic hepatitis C. This group is in turn at high risk for serious liver disease such as liver cirrhosis, hepatocellular carcinoma and terminal liver disease leading to death.
HCV is an enveloped positive-strand RNA virus. The single strand HCV RNA genome is approximately 9500 nucleotides in length and has a single open reading frame (ORF) encoding a single large polyprotein of about 3000 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce structural and non-structural (NS) proteins, which are components of the mature virus and components involved in replication of the viral genome, respectively (Pawlotsky, 2004). In the case of HCV, the generation of mature nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases. The first one is a metalloprotease located in NS2 that cleaves the NS2-NS3 junction in cis; the second one is a serine protease contained within the N-terminal region of NS3 (henceforth referred to as NS3 protease) and mediates all the subsequent cleavages downstream of NS3, both in cis, at the NS3-NS4A cleavage site, and in trans, at the remaining NS4A-NS4B, NS4B-NS5A, NS5A-NS5B sites. The NS4A protein appears to serve multiple functions, acting as a cofactor for the NS3 protease and possibly assisting in the membrane localization of NS3 and other viral replicase components. The non-structural protein 5A (NS5A) is part of the intracellular membrane-associated viral replication complex (Lindenbach et al., 2005). Mutations in NS5A affect the rate of HCV replication (Blight et al., 2000). NS5A has generated considerable interest because of a postulated role in determining the response to interferon (Pawlotsky, 1999).
Similar to other positive-strand RNA viruses, HCV replication occurs in intimate association with specific intracellular membrane structures, which for HCV has been termed the membranous web (Egger et al., 2002). What host machinery is exploited to establish these sites of viral replication is unknown.
Rab-GTPases are small GTP-binding proteins that regulate vesicular membrane trafficking pathways, behaving as membrane-associated molecular switches (Pfeffer et al., 2004). Rab proteins have been previously implicated in the life cycles of various enveloped viruses being utilized by these viruses for endocytosis, trafficking, and sorting of their proteins (see, e.g., Voderheit et al. 2005). Rab1 has previously been shown to be recruited to a replication complex of an intracellular pathogen (Machner et al. (2006) Developmental cell 11(1), 47; Murata et al. (2006) Nature cell biology 8(9), 971). There, a bacterial protein mimics a GTPase exchange factor that activates Rab1 and recruits it to an organelle that supports bacterial replication, thereby subverting membrane transport from the endoplasmic reticulum. We believe that a similar mechanism might apply for the NS5A-TBC1D20 interaction. Since in this case, the interaction is with a GAP which inactivates Rab 1, the mechanism might involve local inactivation of the Rab1, thereby preventing vesicle transport to the Golgi and promoting their redirection to viral replication sites.
The mechanism by which HCV establishes viral persistence and causes a high rate of chronic liver disease has not been elucidated. Antiviral interventions to date have focused upon, for example, ribavirin and interferon-alpha (IFN-α)-based monotherapy and combination therapy. However, many patients are either not responsive to these therapies, or suffer from relapse after an initial response.
Further methods for identifying anti-HCV agents are of interest in the field.
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